Method of treatment for cancers associated with elevated HER 2 levels

ABSTRACT

Novel methods of treating proliferative disorders characterized by elevated Her-2, and the patient is then administered an effective amount of an HSP90 inhibitor.

This application is a 371 national stage of PCT/US02/16287, filed May23, 2002, and claims the benefit of U.S. Provisional Application No.60/293,246, filed May 23, 2001.

FIELD OF THE INVENTION

The field of the invention relates to chemotherapeutic treatment ofcancers.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art, or relevant, to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

The eukaryotic heat shock protein 90s (HSP90s) are ubiquitous chaperoneproteins that are involved in folding, activation and assembly of a widerange of proteins, including key proteins involved in signaltransduction, cell cycle control and transcriptional regulation.Researchers have reported that HSP90 chaperone proteins are associatedwith important signaling proteins, such as steroid hormone receptors andprotein kinases, including, e.g., Raf-1, EGFR, v-Src family kinases,Cdk4, and ErbB-2 (Buchner J., 1999, TIBS, 24:136-141; Stepanova, L. etal., 1996, Genes Dev. 10: 1491-502; Dai, K. et al., 1996, J. Biol. Chem.271:22030-4). Studies further indicate that certain co-chaperones, e.g.,Hsp70, p60/Hop/Sti1, Hip, Bag1, HSP40/Hdj2/Hsj1, immunophilins, p23, andp50, may assist HSP90 in its function (see, e.g., Caplan, A., 1999,Trends in Cell Biol., 9: 262-68).

Ansamycin antibiotics derived from Streptomyces hygroscopicus are knownto inhibit HSP90s. These antibiotics, e.g., herbimycin A (HA) andgeldanamycin (GM), as well as other HSP90 inhibitors such as radicicol,bind tightly to an N-terminus pocket in HSP90 (Stebbins, C. et al.,1997, Cell, 89:239-250). This pocket is highly conserved and has weakhomology to the ATP-binding site of DNA gyrase (Stebbins, C. et al.,supra; Grenert, J. P. et al., 1997, J. Biol. Chem., 272:23843-50).Further, ATP and ADP have both been shown to bind this pocket with lowaffinity and to have weak ATPase activity (Proromou, C. et al., 1997,Cell, 90: 65-75; Panaretou, B. et al., 1998, EMBO J., 17: 4829-36). Invitro and in vivo studies have demonstrated that occupancy of thisN-terminal pocket by ansamycins and other HSP90 inhibitors alters HSP90function and inhibits protein folding. At high concentrations,ansamycins and other HSP90 inhibitors have been shown to prevent bindingof protein substrates to HSP90 (Scheibel, T., H. et al., 1999, Proc.Natl. Acad. Sci. USA 96:1297-302; Schulte, T. W. et al., 1995, J. Biol.Chem. 270:24585-8; Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci.USA 91:8324-8328). Ansamycins have also been demonstrated to inhibit theATP-dependent release of chaperone-associated protein substrates(Schneider, C., L. et al., 1996, Proc. Natl. Acad. Sci. USA,93:14536-41; Sepp-Lorenzino et al., 1995, J. Biol. Chem.270:16580-16587). In either event, the substrates are degraded by aubiquitin-dependent process in the proteasome (Schneider, C., L., supra;Sepp-Lorenzino, L., et al., 1995, J. Biol. Chem., 270:16580-16587;Whitesell, L. et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 8324-8328).

This substrate destabilization occurs in tumor and non-transformed cellsalike and has been shown to be especially effective on a subset ofsignaling regulators, e.g., Raf (Schulte, T. W. et al., 1997, Biochem.Biophys. Res. Commun. 239:655-9; Schulte, T. W., et al., 1995, J. Biol.Chem. 270:24585-8), nuclear steroid receptors (Segnitz, B., and U.Gehring. 1997, J. Biol. Chem. 272:18694-18701; Smith, D. F. et al.,1995, Mol. Cell. Biol. 15:6804-12), v-src (Whitesell, L., et al., 1994,Proc. Natl. Acad. Sci. USA 91:8324-8328) and certain transmembranetyrosine kinases (Sepp-Lorenzino, L. et al., 1995, J. Biol. Chem.270:16580-16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmann, F.,et al., 1997, Int. J. Cancer 70:221-9; Miller, P. et al., 1994, CancerRes. 54:2724-2730; Mimnaugh, E. G., et al., 1996, J. Biol. Chem.271:22796-801; Schnur, R. et al., 1995, J. Med. Chem. 38:3806-3812). Theansamycin-induced loss of these proteins leads to the selectivedisruption of certain regulatory pathways and results in growth arrestat specific phases of the cell cycle (Muise-Heimericks, R. C. et al.,1998, J. Biol. Chem. 273:29864-72), and apoptsosis, and/ordifferentiation of cells so treated (Vasilevskaya, A. et al., 1999,Cancer Res., 59:3935-40).

Growth arrest of this sort, provided it can be made selective, hasimportant ramifications for the treatment of proliferative diseases,i.e., cancers. Whereas cancer treatments have thus far been limited totraditional surgical removal, radiation, and/or chemotherapy, andwhereas these procedures have been more or less successful, a needremains to develop additional therapies with increased efficacy anddecreased side-effects. There particularly remains a need for cancertreatments that target specific cancer types, e.g., those characterizedby the overexpression of Her-2/neu.

The Her-2/neu oncogene, also called erbB2, encodes a glycoprotein withtyrosine kinase activity known as p185 (Schechter, A. L. et al., 1984,Nature, 312: 513-516). HER-2 is a member of the epidermal growth factorreceptor (EGFR) family and shares partial homology with other familymembers. In normal adult tissues, HER-2 expression is low; however,HER-2 is reported to be overexpressed in many tumors, including asignificant fraction of human tumors of the breast, lung, ovary, andpancreas (Hynes, N. E. and Stem, D. F., 1994, Biochem. Biophys. Acta.,1198: 165-184.) Studies indicate that HER-2 is overexpressed in at least25-30% of breast cancers (McGuire & Greene, 1989, Semin. Oncol. 16:148-155). Furthermore, overexpression of HER-2 in malignant breasttumors is correlated with increased metastasis, chemoresistance and poorsurvival rates (Slamon et al., 1987 Science 235: 177-182).

Because HER-2 overexpression is associated with and contributes to manytypes of mammalian proliferative diseases, a means of controlling suchoverexpression is much needed. A treatment that could simultaneouslyavoid or minimize harm to normal cells and tissues would be mostdesirable. The present invention satisfies these needs and providesrelated advantages as well.

SUMMARY OF THE INVENTION

Applicants have discovered that the sensitivity of cells typical ofvarious proliferative disorders to the ansamycin CNF-101 (“17-AAG”) isproportional to the HER-2 levels in those cells. This finding iscounterintuitive because the vast majority of chemotherapeutic drugsthat target oncogenic proteins display the exact opposite relationshipwith respect to their target proteins usually, elevated levels ofoncogenic target proteins are associated with acquired or innateresistance to a given drug; the more protein, the less sensitivity. See,e.g., Inaba, M., 1997, Nippon Rinsho 55: 1030-1037; Mcleod H et al,1996, Brit. J Cancer 74:508-512; Kinsella, A et al, 1998, Gen.Pharmacol. 30:623-626; Inaba, M., supra; Murakami, Y et al, 2000, Int.J. Oncol. 17:277-283.

In particular, the invention relates to methods of treating patientswith cell proliferative disorders, including cancers, that areassociated with elevated levels of HER-2 expression. The methods involvetesting to determine an elevated level of HER-2, and administering atherapeutically effective amount of a HSP90 inhibitor. In preferredembodiments, the particular disorder is a cancer, particularly a breastcancer.

Accordingly, in a first aspect the method includes providing cells,tissue, or fluid from a patient suspected of having a proliferativedisorder, testing the cells, tissue, or fluid for one or more of thegene copy number of HER-2, the amount of HER-2 mRNA, or the amount ofHER-2 protein. If the level of HER-2 is elevated relative to normalcells, the method further includes administering to the patient apharmaceutically effective amount of an HSP90 inhibitor.

The inhibitor may be an ansamycin or other type of small molecule HSP90inhibitor, e.g., radicicol, or an analog thereof (see, e.g., U.S. Pat.Nos. 5,977,165, 5,650,430, and 5,597, 846). These compounds may besynthetic or natural. Preferred inhibitors include geldanamycin, 17-AAG,herbimycin A, and macbecin. The inhibitor preferably binds theATP-binding site of a HSP90 and has an IC₅₀ that is lower, preferably atleast two-fold lower, more preferably at least five-fold lower, and mostpreferably at least ten-fold lower for cancerous cells than fornoncancerous cells. The inhibitor preferably exhibits an IC₅₀ of about100 nM or less. Other embodiments feature 75 nM or less, 50 nM or less,25 nM or less, 10 nM or less, and 5 nM or less. In preferredembodiments, the inhibitor is 17-AAG.

In the testing step, preferred embodiments include using nucleic acidhybridization or PCR to determine HER-2 gene copy number or the level ofHER-2 messenger RNA. Additionally or alternatively, one may test for thepresence of the HER-2 protein using, e.g., any of a variety ofantibody-based techniques well known in the art. Testing may be done invivo or in vitro (ex vivo), the latter, e.g., by biopsying cells.

Administration of HSP90 inhibitors may be done ex vivo or directly tothe patient, e.g., parenterally, peripherally, or intralesionally.Testing may also be performed to measure the sensitivity of thepatient's cells to one or more HSP90 inhibitors prior to administrationof a particular HSP90 inhibitor as a therapeutic. This testing may bedone on abnormal cells and/or normal cells alike to determineappropriate therapeutic amounts for administration. Positive and/ornegative standards may be used in the testing step(s) to determine HER-2levels and/or to evaluate inhibitor compound sensitivity.

In some embodiments, the methods of the invention may also include oneor more testing steps following HSP90 inhibitor administration in orderto monitor therapeutic effect. This step may include measuring amolecular marker that is indicative of the level of HER-2, e.g., HER-2mRNA, a cyclin, and/or the level of phosphorylated AKT. Cyclins arepreferably selected from the group consisting of D1 and D3, as thosespecies are known in the art. Standards may also be used in this step.

An advantage of the invention is the ability to selectively targetproliferative disorders that are characterized by cells exhibitingelevated HER-2 levels by administering an amount of HSP90 inhibitor thatis effective against these particular cells, but that is relativelyineffective against normal cells. Other advantages will be apparent fromthe figures, the detailed specification, and the claims to follow.

Specifically, in one aspect of the invention there is provided a methodof treating a patient having breast cancer characterized by breastcancer cells that have elevated levels of HER-2 expression as comparedto non-cancerous cells of the same type. The method comprises the stepsof

(a) providing breast cancer cells from said patient;

(b) testing said provided cells for one or more of the gene copy numberof HER-2, the amount of HER-2 mRNA, or the amount of HER-2 protein as anindication of the level of HER-2 expression; and

if said level of HER-2 expression is elevated as compared tonon-cancerous cells of the same type, (c) administering to said patienta pharmaceutically effective amount of an HSP90 inhibitor, wherein saidinhibitor consists essentially of an ansamycin that binds into theATP-binding site of a HSP90. The HSP90 inhibitor has an IC₅₀ at leasttwo-fold lower for said provided cells in said patient having elevatedHer-2 than for cells that do not have elevated Her-2 and theadministered amount is correspondingly lower than the amount that wouldbe required for cells that do not have elevated Her-2. The methodfurther comprises the step of (d) testing to monitor therapeutic effectafter said administration by measuring a molecular marker indicative ofthe level of HER-2.

In some embodiments of this method, the molecular marker is selectedfrom one or more of the group consisting of a cyclin, and phosphorylatedAKT.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the antiproliferative effect of CNF-101 (17-AAG) (17-AAG)on a panel of human breast cancer cells in vitro.

FIG. 2 shows the changes in levels of a HSP90 client protein (Raf-1) andof four downstream target proteins in HER-2+++ and HER-2-cells aftertreatment with CNF-101 (17-AAG) (17-AAG) in vitro.

FIG. 3 shows the changes in protein levels, phosphorylation status, andkinase activity of AKT in HER-2+++ and HER-2-cells after treatment withCNF-101 (17-AAG) (17-AAG) in vitro.

FIG. 4 shows the changes in protein levels and phosphorylation status ofAKT in a panel of breast cancer cell lines after treatment with 1 uMCNF-101 (17-AAG) (17-AAG) in vitro.

FIG. 5 shows the changes in levels of a HSP90 client protein (HER-2) andchanges in protein levels and phosphorylation status of the downstreamtarget protein AKT in HER-2+++ breast carcinoma xenografts at varioustimes after treatment with CNF-101 (17-AAG) (17-AAG) in vivo.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms have the following meanings.

By “elevated levels of HER-2 expression” is meant that more HER-2protein is present relative to normal cells. This may be a consequenceof gene amplification (multiple gene copy), enhanced transcriptionand/or translation from existing HER-2 genes and mRNA, and/or increasedpost-translational stability. By “elevated HER-2” is meant one or moreof elevated HER-2 gene copy, mRNA transcript, or protein.

An “HSP90-inhibiting compound” or “HSP90-inhibitor” is one that disruptsthe structure and/or function of an HSP90 chaperone protein and/or aprotein that is dependent on HSP90. HSP90 proteins are highly conservedin nature (see, e.g., NCBI accession #'s P07900 and XM 004515 (humanαand β HSP90, respectively), P11499 (mouse), AAB2369 (rat), P46633(chinese hamster), JC1468 (chicken), AAF69019 (flesh fly), AAC21566(zebrafish), AAD30275 (salmon), 002075 (pig), NP 015084 (yeast), andCAC29071 (frog). Grp94 and Trap-1 are related molecules falling withinthe definition of an HSP90-as used herein. There are thus many differentHSP90s, all with anticipated similar effect and inhibition capabilities.The HSP90 inhibitors of the invention may be specifically directedagainst an HSP90 of the specific host patient or may be identified basedon reactivity against an HSP90 homolog from a different species, or anHSP90 variant. The inhibitors used may be ring-structured antibiotics,e.g., benzoquinone ansamycins, or other types of molecules, e.g.,antisense nucleic acids, or molecules such as radicicol and analogsthereof.

An “ansamycin” includes but is not limited to geldanamycin, 17-AAG,herbimycin A, and macbecin. The specific ansamycin 17-AAG stands for17-allylamino-17-demethoxygeldanamycin. These and other ansamycins thatcan be used are well-known in the art. See, e.g., U.S. Pat. Nos.3,595,955, 4, 261, 989, 5,387,584, and 5,932,566. Ansamycins may besynthetic, naturally-occurring, or else derivatives of naturallyoccurring ansamycins that are prepared using standard chemicalderivatization techniques.

A “pharmaceutically effective amount” means an amount which is capableof providing a therapeutic or prophylactic effect. The specific dose ofcompound administered according to this invention to obtain therapeuticand/or prophylactic effect will, of course, be determined by theparticular circumstances surrounding the case, including, for example,the specific compound administered, the route of administration, thecondition being treated, and the individual being treated. A typicaldaily dose (administered in single or divided doses) will contain adosage level of from about 0.01 mg/kg to about 100 and more preferaby 50mg/kg of body weight of an active compound of this invention. Preferreddaily doses generally will be from about 0.05 mg/kg to about 20 mg/kgand ideally from about 0.1 mg/kg to about 10 mg/kg.

The preferred therapeutic effect is the inhibition, to some extent, ofthe growth of cells characteristic of a proliferative disorder, e.g.,breast cancer. A therapeutic effect will also normally, but need not,relieve to some extent one or more of the symptoms other than cellgrowth or size of cell mass. A therapeutic effect may include, forexample, one or more of 1) a reduction in the number of cells; 2) areduction in cell size; 3) inhibition (i.e., slowing to some extent,preferably stopping) of cell infiltration into peripheral organs, e.g.,in the instance of cancer metastasis; 3) inhibition (i.e., slowing tosome extent, preferably stopping) of tumor metastasis; 4) inhibition, tosome extent, of cell growth; and/or 5) relieving to some extent one ormore of the symptoms associated with the disorder.

The term “IC₅₀” is defined as the concentration of an HSP90 inhibitorrequired to achieve killing of 50% of the cells of a population, or of aparticular cell type, e.g., cancerous versus noncancerous cells within agreater cell population. The IC₅₀ is preferably, although notnecessarily, greater for normal cells than for cells exhibiting aproliferative disorder.

By a “standard” is meant a positive or negative control. A negativecontrol in the context of HER-2 expression levels is, e.g., a samplepossessing an amount of HER-2 (gene, transcript, and/or correspondingprotein product) that correlates with a normal cell. A negative controlmay also include a sample that contains no HER-2 gene or gene-product,e.g., mRNA or protein. By contrast, a positive control does contain aHER-2 gene or gene product, preferably of an amount that correlates withoverexpression as found in proliferative disorders, e.g., breastcancers. The controls may be from cell or tissue samples, or elsecontain purified ligand (or absent ligand), immobilized or otherwise. Insome embodiments, one or more of the controls may be in the form of adiagnostic “dipstick.”

By “selectively targeting” is meant affecting one type of cell to agreater extent than another, e.g., in the case of cells with high asopposed to relatively low or normal Her-2 levels.

General

The present invention concerns methods for treating cell proliferativedisorders associated with high HER-2 levels based on the observationthat cells which overexpress HER-2 are more sensitive to HSP90inhibitors than cells which do not overexpress HER-2, e.g., normalcells. In any event, for diagnosis prior to treatment, and inembodiments where post-treatment evaluation is made, HER-2 levels may bedetermined.

Determining HER-2 Levels

Many different types of methods are known in the art for determiningprotein concentrations and measuring or predicting the level of proteinswithin cells and in fluid samples. Indirect techniques include nucleicacid hybridization and amplification using, e.g., polymerase chainreaction (PCR). These techniques are known to the person of skill andare discussed, e.g., in Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., Ausubel, et al., Current Protocols inMolecular Biology, John Wiley & Sons, NY, 1994, and, as specificallyapplied to the quantification, detection, and relative activity ofHer-2/neu inpatient samples, e.g., in U.S. Pat. Nos. 4,699,877,4,918,162, 4,968,603, and 5,846,749. A brief discussion of two generictechniques that can be used follows.

a. Immunodetection

The determination of whether cells overexpress or contain elevatedlevels of HER-2 can be determined using techniques well known in theart, e.g., antibody techniques such as immunoblotting,radioimmunoassays, western blotting, immunoprecipitation, enzyme-linkedimmunosorbant assays (ELISA), and derivative techniques that make use ofantibodies directed against HER-2. As an example, HER-2 expression inbreast cancer cells can be determined with the use of animmunohistochemical assay, such as the Dako Hercep™ test (Dako Corp.,Carpinteria, Calif.). The Hercep™ test is an antibody staining assaydesigned to detect HER-2 overexpression in tumor tissue specimens. Thisparticular assay grades HER-2 expression into four levels: 0, 1, 2, and3, with level 3 representing the highest level of HER-2 expression.Accurate quantitation can be enhanced by employing an Automated CellularImaging System (ACIS) as described, e.g., by Press, M, et al, 2000,Modern Pathology 13:225A.

Antibodies, polyclonal or monoclonal, can be purchased from a variety ofcommercial suppliers, or may be manufactured using well-known methods,e.g., as described in Harlow et al., Antibodies: A Laboratory Manual,2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1988).

b Nucleic Acid Techniques

i. PCR

HER-2 overexpression can also be determined at the nucleic acid levelsince there is a reported high correlation between overexpression of theHER-2 protein and amplification of the gene that codes for it. One wayto test this is by using RT-PCR. The genomic and cDNA sequences forHER-2 are known. Specific DNA primers can be generated using standard,well-known techniques, and can then be used to amplify template alreadypresent in the cell. An example of this is described in Kurokawa, H etal, Cancer Res. 60: 5887-5894 (2000), (which describes a forward primerhaving sequence 5′-TCTGGACGTGCCAGTGTGAA-3′ (SEQ ID NO. 1) and a reverseprimer having sequence 5′-TGCTCCCTGAGGACACATCA-3′ (SEQ ID NO. 2)). PCRcan be standardized such that quantitative differences are observed asbetween normal and abnormal cells, e.g., cancerous and noncancerouscells. Well known methods employing, e.g., densitometry, can be used toquantitate and/or compare nucleic acid levels amplified using PCR.

ii. Hybridization-based Techniques

Similarly, fluorescent in situ hybridization (FISH) assays and otherassays can be used, e.g., Northern and/or Southern blotting. These relyon nucleic acid hybridization between the HER-2 gene or mRNA and acorresponding nucleic acid probe that can be designed in the same or asimilar way as for PCR primers, above. See, e.g., Mitchell M S, andPress M F., 1999, Semin. Oncol., Suppl. 12:108-16. For FISH, thisnucleic acid probe can be conjugated to a fluorescent molecule, e.g.,fluorescein and/or rhodamine, that preferably does not interfere withhybridization, and which fluorescence can later be measured followinghybridization. See, e.g., Kurokawa, H et al, Cancer Res. 60: 5887-5894(2000) (describing a nucleic acid probe having sequence 5′-FAM-CAGAAGGCCAAGTCCGCAGAAGCC-TAMRA-p-3′ (SEQ ID NO. 3)). ACIS-basedapproaches as described above can be employed to make the assay morequantitative (de la Torre-Bueno, J, et al, 2000, Modern Pathology13:221A).

EXAMPLE 1 Antiproliferative Effects of 17-AAG (CNF-101) on Cancer CellsWhich Express Different Amounts of HER-2

Human cancer cell lines BT-474, SKBR-3, SKOV-3, MCF-Her2, MCF-7,MDA-468, MDA-231, T47-D, and MDA435 were obtained from the American TypeCulture Collection (“ATCC;” Manassas, Va., USA). Cell lines weremaintained in DMEM/F2/1 medium, supplemented with 5% fetal calf serum(BRL), 2 mM glutamine, and 50 U/ml each of penicillin and streptomycin,at 37° C. in 5% CO₂.

Cells were treated for 24 hours with CNF-101 (17-AAG) at concentrationsof 1 to 1000 nM. Following treatment, the nuclei were stained withethidium bromide and analyzed by flow cytometry. Nuclei were isolatedfor flow cytometry assays, stained with ethidium bromide and analyzedusing a Becton Dickinson fluorescence-activated cell sorter. Statisticaldata was obtained using Multicycle program software.

As shown in FIG. 1, in the cell lines examined, CNF-101 (17-AAG)treatment caused growth arrest in all the cell lines tested. Those celllines expressing the highest levels of HER-2 were the most sensitive toCNF-101 (17-AAG) (FIG. 1). FIG. 1 shows the antiproliferative effect ofCNF-101 (17-AAG) on a panel of human breast cancer cells in vitro. Thecell lines expressing high endogenous levels of HER-2 (BT-474, SKBR-3,SKOV-3, MDA435) are markedly more sensitive to CNF-101 (17-AAG) than arethe low HER-2 lines (MDA-468, MDA-231, T47D). Furthermore, the 10-folddifference in sensitivity between two cells from a congenic pair (MCF-7vs. MCF-7/HER-2) differing only in their HER-2 levels demonstrate thatHER-2 is the critical factor, rather than other potential variationsbetween the cell lines.

EXAMPLE 2 Changes in Protein Expression Induced by CNF-101 (17-AAG) inHER-2 Negative Cells and Cells Which Overexpress HER-2

Levels of mitotic cyclin expression, HER-2 expression, AKT expressionand Raf1 expression in MCF-7 (HER-2 negative) or BT-474 (HER-2+++) celllines treated with CNF-101 (17-AAG) were assessed using immunoblotanalysis. Immunoblot analysis of lysates from cells treated with either50 nM or 500 nM CNF-101 (17-AAG) were analyzed by Western blot usinganti-cyclin D1, anti-cyclin D3, anti-cyclin cdk4, anti-AKT or anti-Raf1antibodies. All antibodies were obtained from Santa Cruz Biotechnology(Santa Cruz, Calif., USA). Treated cells were harvested, washed with PBSand lysed in NP40 lysis buffer (50 mM Tris pH7.4, 1% NP40,150 mM NaCl,40 mM NaF. 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, and 10 μg/mleach of leupeptin, aprotinin and soybean trypsin inhibitor) for 30 minon ice. Lysates were centrifuged at 15,000×g for 10 min and proteinconcentration determined by bicinchoninic acid protein assay (Pierce).Equal amounts of total protein were resolved by SDS-PAGE and transferredonto Immobilon PVDF membranes (Millipore) by electroblotting. Blots wereblocked overnight in 5% nonfat milk in TBS-T (0.1% Tween-20 TBS, 10 mMTris pH 7.4, 150 mM NaCl) at 4° C. and subsequently probed with eitheranti-HER-2, anti-cylin D1, anti-cyclin D3, anti cdk4, anti-AKT oranti-Raf1 antibodies. Following incubation with HRP-conjugated secondaryantibodies, proteins were detected by chemiluminescence (Amersham).

FIG. 2 shows the changes in levels of HER-2 after treatment with CNF-101(17-AAG) in vitro. Treatment of BT-474 cells (which over express HER-2)with 17-AAG resulted in the rapid decrease in cyclin D1 and cyclin D3(FIG. 2). This decrease in cyclin D1 and D3 protein levels were not seenin MCF-7 cell lines (low HER-2 expression) (FIG. 2). The protein levelsof cdk4, and AKT were not affected in either HER-2 overexpressing cellsor HER low expressing cells after treatment with 17-AAG (FIG. 2). CyclinD1 and cyclin D3 can be used according to the invention to indirectlymonitor HER-2 expression and inhibitory effects thereon induced by HSP90inhibitors.

EXAMPLE 3 Effect of CNF-101 (17-AAG) on AKT Expression, Phosphorylationand Activity

FIG. 3 shows the changes in protein levels, phosphorylation status andkinase activity of AKT in HER-2+++ and HER-2-cells after treatment withCNF-101 (17-AAG) in vitro. Cells were treated with 1 uM CNF-101 (17-AAG)for 24 hrs, washed, harvested and lysed in NP-40. Lysates were resolvedby SDS-PAGE, transferred onto Immobilon PVDF membranes, blocked in 5%nonfat milk and probed with antibodies to AKT or tyrosine phosphorylatedAKT (phospho-AKT) from Upstate. Following incubation with HRP-conjugatedsecondary antibodies, proteins were detected using chemiluminescnce(Amersham). AKT kinase activity was assessed followingimmunopreciptation by a standard kinase assay with the recombinant AKTsubstrate protein glycogen synthase kinase-1 (GSK-1) and 32-P labelledATP. AKT is a key kinase in tumor cells. The protein itself is not aHSP90 client, so treatment with CNF-101 (17-AAG) does not causesignificant loss of the protein. However, because the essential upstreamregulator of the AKT pathway in breast cancer (i.e. HER-2) is degraded,the signals necessary for AKT phosphorylation (and hence activity of theprotein) are lost, so phospho-AKT disappears from the treated cells. Theexperiment also shows a quantitative relationship between HER-2 levelsand AKT inhibition: 100 nM CNF-101 (17-AAG) causes complete loss of AKTactivity in high HER-2 (SKBr3) cells within 3 hrs, but even ten timesthat amount requires 12 hours to cause even partial loss of AKT kinaseactivity in the HER-2-low MCF-7 line.

EXAMPLE 4 Effect of CNF-101 (17-AAG) on Levels of AKT and PhosphorylatedAKT in a Panel of Human Cell Lines

FIG. 4 shows the changes in protein levels and phosphorylation status ofAKT in a panel of breast cancer cell lines after treatment with 1 uMCNF-101 (17-AAG) in vitro. Cells were treated with 1 uM CNF-101 (17-AAG)for 24 hrs, washed, harvested and lysed in NP-40. Lysates were resolvedby SDS-PAGE, transferred onto Immobilon PVDF membranes, blocked in 5%nonfat milk and probed with antibodies to AKT or tyrosine phosphorylatedAKT (phospho-AKT) from Upstate. Following incubation with HRP-conjugatedsecondary antibodies, proteins were detected using chemiluminescnce(Amersham). AKT is a key kinase in tumor cells. The protein itself isnot a HSP90 client, so treatment with CNF-101 (17-AAG) does not causesignificant loss of the protein. However, because the essential upstreamregulator of the AKT pathway in breast cancer (i.e. HER-2) is degraded,the signals necessary for AKT phosphorylation (and hence activity of theprotein) are lost, so phospho-AKT disappears from the treated cells.This experiment demonstrates that CNF-101-induced degradation of HER-2causes loss of AKT activity in multiple cell lines, but that those lineswith high HER-2 (BT-474, SKBR-3) display a much more rapid and completeloss of AKT activity than do those lines with low HER-2 expression (U87,MCF-7, MDA-468)

EXAMPLE 5 Studies on Breast Carcinoma Xenografts

FIG. 5 shows the changes in levels of a HSP90 client protein (HER-2) andchanges in protein levels and phosphorylation status of the downstreamtarget protein AKT in HER-2+++ breast carcinoma xenografts at varioustimes after treatment with CNF-101 (7-AAG) in vivo. BT-474 breast tumorcells were implanted subcutaneously on the flank of Swiss nu/nu mice andallowed to become established to a diameter of 5 mm. Animals weretreated intraperitoneally with optimal doses of CNF-101 (17-AAG) andtumors removed at the indicated times thereafter. Tumor tissue wassnap-frozen at the time of removal. For analysis, tissues were rapidlythawed, lysed and assaysed for HER-2, AKT and phospho-AKT by Westernblotting. This experiment demonstrates that the CNF-101-induceddegradation of HER-2 and loss of AKT activity is not merely an in vitrophenomenon, but that the drug can induce these key mechanism-basedchanges in a whole animal setting with the same potency and kinetics.

To summarize Examples 2-5, addition of CNF-101 (17-AAG) to culturedcells results in antiproliferative effects that are strongest in cellsthat overexpress HER-2. For example, in cell lines that expressdifferent amounts of HER-2 (BT-174; SKBR-3>SK−OV-3>MCF-HER-2>MDA-435>MCF-7; MDA-231; MDA-468>T47-D), cells expressinghigher amounts of HER-2 were more sensitive to the HSP90 inhibitor,CNF-101 (17-AAG), than cells expressing low amounts of HER-2 (See FIG.1).

Western blot analysis of protein expression in cells overexpressingHER-2 (BT-474) and in cells which are HER-2 low (e.g., MCF-7) aftertreatment with either 50 nM or 500 nM CNF-101 (17-AAG) at 0, 4, 12, and24 hours indicated that treatment of cells overexpressing HER-2 resultedin a decrease in cyclin-D1 and cyclin-D3 protein expression (FIG. 2).This decrease was not seen when HER-2 negative cells were treated withCNF-101 (17-AAG), whereas the drug did cause degradation of the HSP90client protein Raf-1 in both cell types (FIG. 2).

Further, treatment of cells which overexpress HER-2 with CNF-101(17-AAG) resulted in rapid loss of AKT kinase activity and amount ofphosphorylated (=active) AKT (FIGS. 3 and 4). This rapid loss of AKTactivity and phosphorylated AKT was not seen in cells which express lowamounts of HER-2 (FIGS. 3 and 4). The effect of CNF-101 on thephosphorylation level and activity of AKT correlates with the data shownin FIG. 1, which shows that cells which overexpress HER-2 are moresensitive to HSP90 inhibitors than cells which express low levels ofHER-2. Similarly, those cells which express the highest amounts of HER-2showed the most rapid loss of AKT phosphorylation, suggesting that theinhibition of cell proliferation shown in FIG. 1 is due todown-regulation of AKT activity, and that the loss of AKT activity isitself induced by the degradation of HER-2.

Analysis of protein expression and AKT phosphorylation levels of ofBT-474 Xenografts treated with 50 mg/kg CNF-101 (17-AAG), showed thatHER-2 protein levels declined, AKT protein levels remained constant, butthat the amount of phosphorylated (“active”) AKT protein declinedrapidly (FIG. 5). These data demonstrate that the effects of CNF-101(17-AAG) shown in vitro are also valid in vivo.

Example 6 Formulation and Administration of Pharmaceutical Compositions

Geldanamycin may be prepared according to U.S. Pat. No. 3,595,955 usingthe subculture of Streptomyces hygroscopicus that is on deposit with theU.S. Department of Agriculture, Northern Utilization and ResearchDivision, Agricultural Research, Peoria, Ill., USA, accession numberNRRL 3602. Numerous derivatives of this compound may be fashioned asspecified in U.S. Pat. Nos. 4,261,989, 5,387,584, and 5,932,566,according to standard techniques.

Those of ordinary skill in the art are familiar with formulation andadministration techniques that can be employed in use of the invention,e.g., as discussed in Goodman and Gilman's, The Pharmacological Basis ofTherapeutics, current edition; Pergamon Press; and Remington'sPharmaceutical Sciences (current edition.) Mack Publishing Co., Easton,Pa.

The compounds utilized in the methods of the instant invention may beadministered either alone or in combination with pharmaceuticallyacceptable carriers, excipients or diluents, in a pharmaceuticalcomposition, according to standard pharmaceutical practice. Thecompounds can be administered orally or parenterally, including theintraventous, intramuscular, intraperitoneal, subcutaneous, rectal andtopical routes of administration.

For example, the therapeutic or pharmaceutical compositions of theinvention can be administered locally to the area in need of treatment.This may be achieved by, for example, but not limited to, local infusionduring surgery, topical application, e.g., cream, ointment, injection,catheter, or implant, said implant made, e.g, out of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers. The administration can also be by directinjection at the site (or former site) of a tumor or neoplastic orpre-neoplastic tissue.

Still further, the therapeutic or pharmaceutical composition can bedelivered in a vesicle, e.g., a liposome (see, for example, Langer,1990, Science, 249:1527-1533; Treat et al., 1989, Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Bernstein and Fidler(eds.), Liss, N.Y., pp. 353-365).

The pharmaceutical compositions used in the methods of the presentinvention can be delivered in a controlled release system. In oneembodiment, a pump may be used (see, Sefton, 1987, CRC Crit. Ref.Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery, 88:507; Saudek etal., 1989, N. Engl. J. Med., 321:574). Additionally, a controlledrelease system can be placed in proximity of the therapeutic target.(see, Goodson, 1984, Medical Applications of Controlled Release, Vol. 2,pp. 115-138).

The pharmaceutical compositions used in the methods of the instantinvention can contain the active ingredient in a form suitable for oraluse, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups or elixirs. Compositions intended for oral use maybe prepared according to any method known to the art for the manufactureof pharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tabletscontain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,such as microcrystalline cellulose, sodium crosscarmellose, corn starch,or alginic acid; binding agents, for example starch, gelatin,polyvinyl-pyrrolidone or acacia, and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to mask the taste of the drug ordelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, awater soluble taste masking material such ashydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delaymaterial such as ethyl cellulose, or cellulose acetate butyrate may beemployed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions used in the methods of the instantinvention may also be in the form of an oil-in-water emulsions. The oilyphase may be a vegetable oil, for example olive oil or arachis oil, or amineral oil, for example liquid paraffin or mixtures of these. Suitableemulsifying agents may be naturally-occurring phosphatides, for examplesoy bean lecithin, and esters or partial esters derived from fatty acidsand hexitol anhydrides, for example sorbitan monooleate, andcondensation products of the said partial esters with ethylene oxide,for example polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening, flavoring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solutionthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

The HSP90 inhibitors used in the methods of the present invention mayalso be administered in the form of suppositories for rectaladministration of the drug. These compositions can be prepared by mixingthe inhibitors with a suitable non-irritating excipient which is solidat ordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter, glycerinated gelatin, hydrogenated vegetable oils,mixtures of polyethylene glycols of various molecular weights and fattyacid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing an HSP90 inhibitor can be used. As used herein, topicalapplication can include mouth washes and gargles.

The compounds used in the methods of the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles and delivery devices, or via transdermal routes, using thoseforms of transdermal skin patches well known to those of ordinary skillin the art. To be administered in the form of a transdermal deliverysystem, the dosage administration will, of course, be continuous ratherthan intermittent throughout the dosage regimen.

The methods and compounds of the instant invention may also be used inconjunction with other well known therapeutic agents that are selectedfor their particular usefulness against the condition that is beingtreated. For example, the instant compounds may be useful in combinationwith known anti-cancer and cytotoxic agents. Further, the instantmethods and compounds may also be useful in combination with otherinhibitors of parts of the signaling pathway that links cell surfacegrowth factor receptors to nuclear signals initiating cellularproliferation.

The methods of the present invention may also be useful with otheragents that inhibit angiogenesis and thereby inhibit the growth andinvasiveness of tumor cells, including, but not limited to VEGF receptorinhibitors, including ribozymes and antisense targeted to VEGFreceptors, angiostatin and endostatin.

Examples of antineoplastic agents, which can be used in combination withthe methods of the present invention include, in general, alkylatingagents, anti-metabolites; epidophyllotoxin; an antineoplastic enzyme; atopoisomerase inhibitor; procarbazine; mitoxantrone; platinumcoordination complexes; biological response modifiers and growthinhibitors; hormonal/anti-hormonal therapeutic agents and haematopoieticgrowth factors.

Example classes of antineoplastic agents include, for example, theanthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the epothilones, discodermolide,the pteridine family of drugs, diynenes and the podophyllotoxins.Particularly useful members of those classes include, for example,carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin orpodo-phyllotoxin derivatives such as etoposide, etoposide phosphate orteniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine,leurosine, paclitaxel and the like. Other useful antineoplastic agentsinclude estramustine, carboplatin, cyclophosphamide, bleomycin,gemcitibine, ifosamide, melphalan, hexamethyl melamine, thiotepa,cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase,camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide,leuprolide, pyridobenzoindole derivatives, interferons and interleukins.

When a HSP90 inhibitor used in the methods of the present invention isadministered into a human subject, the daily dosage will normally bedetermined by the prescribing physician with the dosage generallyvarying according to the age, weight, and response of the individualpatient, as well as the severity of the patient's symptoms.

In one exemplary application, a suitable amount of a HSP90 inhibitor isadministered to a mammal undergoing treatment for cancer, for example,breast cancer. Administration occurs in an amount of each type ofinhibitor of between about 0.1 mg/kg of body weight to about 60 mg/kg ofbody weight per day, preferably of between 0.5 mg/kg of body weight toabout 40 mg/kg of body weight per day. A particular therapeutic dosagethat comprises the instant composition includes from about 0.01 mg toabout 1000 mg of a HSP90 inhibitor. Preferably, the dosage comprisesfrom about 1 mg to about 1000 mg of a HSP90 inhibitor.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., an effectiveamount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 0.1 mg to 1000 mg, preferably from about 1mg to 300 mg, more preferably 10 mg to 200 mg, according to theparticular application.

The actual dosage employed may be varied depending upon the requirementsof the patient and the severity of the condition being treated.Determination of the proper dosage for a particular situation is withinthe skill of the art. Generally, treatment is initiated with smallerdosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

The amount and frequency of administration of the HSP90 inhibitors usedin the methods of the present invention and if applicable otherchemotherapeutic agents and/or radiation therapy will be regulatedaccording to the judgment of the attending clinician (physician)considering such factors as age, condition and size of the patient aswell as severity of the disease being treated.

The chemotherapeutic agent and/or radiation therapy can be administeredaccording to therapeutic protocols well known in the art. It will beapparent to those skilled in the art that the administration of thechemotherapeutic agent and/or radiation therapy can be varied dependingon the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

Also, in general, the HSP90 inhibitor and the chemotherapeutic agent donot have to be administered in the same pharmaceutical composition, andmay, because of different physical and chemical characteristics, have tobe administered by different routes. For example, the HSP90 inhibitormay be administered orally to generate and maintain good blood levelsthereof, while the chemotherapeutic agent may be administeredintravenously. The determination of the mode of administration and theadvisability of administration, where possible, in the samepharmaceutical composition, is well within the knowledge of the skilledclinician. The initial administration can be made according toestablished protocols known in the art, and then, based upon theobserved effects, the dosage, modes of administration and times ofadministration can be modified by the skilled clinician.

The particular choice of HSP90 inhibitor, and chemotherapeutic agentand/or radiation will depend upon the diagnosis of the attendingphysicians and their judgment of the condition of the patient and theappropriate treatment protocol.

The HSP90 inhibitor, and chemotherapeutic agent and/or radiation may beadministered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the proliferative disease, the condition ofthe patient, and the actual choice of chemotherapeutic agent and/orradiation to be administered in conjunction (i.e., within a singletreatment protocol) with the HSP90 inhibitor.

If the HSP90 inhibitor, and the chemotherapeutic agent and/or radiationare not administered simultaneously or essentially simultaneously, thenthe initial order of administration of the HSP90 inhibitor, and thechemotherapeutic agent and/or radiation, may not be important. Thus, theHSP90 inhibitor may be administered first followed by the administrationof the chemotherapeutic agent and/or radiation; or the chemotherapeuticagent and/or radiation may be administered first followed by theadministration of the HSP90 inhibitor. This alternate administration maybe repeated during a single treatment protocol. The determination of theorder of administration, and the number of repetitions of administrationof each therapeutic agent during a treatment protocol, is well withinthe knowledge of the skilled physician after evaluation of the diseasebeing treated and the condition of the patient. For example, thechemotherapeutic agent and/or radiation may be administered first,especially if it is a cytotoxic agent, and then the treatment continuedwith the administration of the HSP90 inhibitor followed, wheredetermined advantageous, by the administration of the chemotherapeuticagent and/or radiation, and so on until the treatment protocol iscomplete.

Thus, in accordance with experience and knowledge, the practicingphysician can modify each protocol for the administration of a component(therapeutic agent-i.e., HSP90 inhibitor, chemotherapeutic agent orradiation) of the treatment according to the individual patient's needs,as the treatment proceeds.

The attending clinician, in judging whether treatment is effective atthe dosage administered, will consider the general well-being of thepatient as well as more definite signs such as relief of disease-relatedsymptoms, inhibition of tumor growth, actual shrinkage of the tumor, orinhibition of metastasis. Size of the tumor can be measured by standardmethods such as radiological studies, e.g., CAT or MRI scan, andsuccessive measurements can be used to judge whether or not growth ofthe tumor has been retarded or even reversed. Relief of disease-relatedsymptoms such as pain, and improvement in overall condition can also beused to help judge effectiveness of treatment.

The foregoing examples are not limiting and are merely representative ofvarious aspects and features of the present invention. All documentscited are indicative of the levels of skill in the art to which theinvention pertains. The disclosure of each document is incorporated byreference herein to the same extent as if each had been incorporated byreference in its entirety individually, although none of the documentsis admitted to be prior art.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described illustrate preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Certain modifications and other uses will occur to thoseskilled in the art, and are encompassed within the spirit of theinvention, as defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention withoutdeparting from the scope and spirit of the invention. Thus, suchadditional embodiments are within the scope of the invention and thefollowing claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention in the use of such teinis andexpressions of excluding any equivalents of the features shown anddescribed, or portions thereof. It is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modifications and variations of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group, and exclusions of individual members asappropriate.

Other embodiments are within the following claims.

1. A method of treating a patient having breast cancer characterized bybreast cancer cells that have elevated levels of HER-2 expression ascompared to non-cancerous cells of the same type, comprising: providingbreast cancer cells from said patient; testing said provided cells forone or more of the gene copy number of HER-2 the amount of HER-2 mRNA,or the amount of HER-2 protein as an indication of the level of HER-2expression; and if said level of HER-2 expression is elevated ascompared to non-cancerous cells of the same type, administering to saidpatient a pharmaceutically effective amount of an HSP90 inhibitor,wherein said HSP90 inhibitor has an IC₅₀ at least two-fold lower forsaid provided cells in said patient having elevated Her-2 than for cellsthat do not have elevated Her-2 and the administered amount iscorrespondingly lower than the amount that would be required for cellsthat do not have elevated Her-2, and wherein said method of treating apatient further comprises testing to monitor therapeutic effect aftersaid administration by measuring a molecular marker indicative of thelevel of HER-2 wherein said inhibitor consists essentially of anansamycin that binds into the ATP-binding site of a HSP90.
 2. The methodof claim 1 wherein said testing comprises using nucleic acidhybridization or PCR to determine the level of HER-2 messenger RNA. 3.The method of claim 1 wherein said testing comprises the use ofantibodies to determine the level of HER-2.
 4. The method of claim 1wherein said testing is done ex vivo.
 5. The method of claim 1 whereinsaid administering is intralesional.
 6. The method of claim 1 whereinsaid administering is parenteral.
 7. The method of claim 1 wherein saidHSP90 inhibitor has an IC₅₀ at least five-fold lower for said cells insaid patient having elevated Her-2 than for cells that do not haveelevated Her-2.
 8. The method of claim 1 wherein said HSP90 inhibitorhas an IC₅₀ at least ten-fold lower for said cells in said patienthaving elevated Her-2 than for cells that do not have elevated Her-2. 9.The method of claim 1 further comprising testing cells of said patientfor sensitivity to HSP90 inhibitors prior to administration of saidHSP90 inhibitor.
 10. The method of claim 1 wherein said testingcomprises the use of one or more standards.
 11. The method of claim 1wherein said inhibitor exhibits an IC₅₀ of about 100 nM or less in saidcells having elevated Her-2.
 12. The method of claim 1 wherein saidinhibitor exhibits an IC₅₀ of about 75 nM or less in said cells havingelevated Her-2.
 13. The method of claim 1 wherein said inhibitorexhibits an IC₅₀ of about 50 nM or less in said cells having elevatedHer-2.
 14. The method of claim 1 wherein said inhibitor exhibits an IC₅₀of about 25 nM or less in said cells having elevated Her-2.
 15. Themethod of claim 1 wherein said inhibitor exhibits an IC₅₀ of about 10 nMor less in said cancerous cells.
 16. The method of claim 1 wherein saidinhibitor exhibits an IC₅₀ of about 5 nM or less in said cells havingelevated Her-2.
 17. A method of treating a patient having breast cancercharacterized by breast cancer cells that have elevated levels of HER-2expression as compared to non-cancerous cells of the same type,comprising: providing breast cancer cells from said patient; testingsaid provided cells for one or more of the gene copy number of HER-2 theamount of HER-2 mRNA, or the amount of HER-2 protein as an indication ofthe level of HER-2 expression; and if said level of HER-2 expression iselevated as compared to non-cancerous cells of the same type,administering to said patient a pharmaceutically effective amount of anHSP90 inhibitor, wherein said HSP90 inhibitor has an IC₅₀ at leasttwo-fold lower for said provided cells in said patient having elevatedHer-2 than for cells that do not have elevated Her-2 and theadministered amount is correspondingly lower than the amount that wouldbe required for cells that do not have elevated Her-2, and wherein saidmethod of treating a patient further comprises testing to monitortherapeutic effect after said administration by measuring a molecularmarker indicative of the level of HER-2 wherein said inhibitor is anansamycin that binds into the ATP-binding site of a HSP90, wherein saidmolecular marker is selected from one or more of the group consisting ofa cyclin, and phosphorylated AKT.
 18. The method of claim 17 whereinsaid molecular marker is a cyclin selected from the group consisting ofD1 and D3.
 19. The method of claim 1, wherein said cells that do nothave elevated Her-2 are from said patient.
 20. The method of claim 1,wherein said IC₅₀ value is calculated using one or more standards. 21.The method of claim 1 wherein said elevated level is at least two-foldgreater for said cells in said patient having elevated Her-2 than forcells that do not have elevated Her-2.
 22. The method of claim 1 whereinsaid elevated level is at least three-fold greater for said cells insaid patient having elevated Her-2 than for cells that do not haveelevated Her-2.
 23. The method of claim 1 wherein said elevated level isat least five-fold greater for said cells in said patient havingelevated Her-2 than for cells that do not have elevated Her-2.
 24. Themethod of claim 1 wherein said elevated level is at least ten-foldgreater for said cells in said patient having elevated Her-2 than forcells that do not have elevated Her-2.
 25. The method of claim 1 whereinsaid elevated level is at least twenty five-fold for said cells in saidpatient having elevated Her-2 than for cells that do not have elevatedHer-2.
 26. The method of claim 1, wherein said ansamycin is selectedfrom the group consisting of geldanamycin, 17-AAG, herbimycin A, andmacbecin.
 27. The method of claim 1, wherein said inhibitor is 17-AAG.28. A method of treating a patient having breast cancer characterized bybreast cancer cells that have elevated levels of HER-2 expression ascompared to non-cancerous cells of the same type, comprising: providingbreast cancer cells from said patient; testing said provided cells forthe gene copy number of HER-2; and if said gene copy number is elevatedas compared to non-cancerous cells of the same type, administering tosaid patient a pharmaceutically effective amount of an HSP90 inhibitor,wherein said HSP90 inhibitor has an IC₅₀ at least two-fold lower forsaid provided cells in said patient having elevated Her-2 than for cellsthat do not have elevated Her-2 and the administered amount iscorrespondingly lower than the amount that would be required for cellsthat do not have elevated Her-2, and wherein said method of treating apatient further comprises testing to monitor therapeutic effect aftersaid administration by measuring a molecular marker indicative of thelevel of HER-2 wherein said inhibitor consists essentially of anansamycin that binds into the ATP-binding site of a HSP90.
 29. Themethod of claim 1, wherein said testing to determine the HER-2 gene copynumber is done using a Southern blot technique.